EP0754764A2 - Procédé et réactif pour la détermination spécifique d'ARNm - Google Patents

Procédé et réactif pour la détermination spécifique d'ARNm Download PDF

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Publication number
EP0754764A2
EP0754764A2 EP96111586A EP96111586A EP0754764A2 EP 0754764 A2 EP0754764 A2 EP 0754764A2 EP 96111586 A EP96111586 A EP 96111586A EP 96111586 A EP96111586 A EP 96111586A EP 0754764 A2 EP0754764 A2 EP 0754764A2
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EP
European Patent Office
Prior art keywords
mixture
buffer
sample
reaction vessel
coated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP96111586A
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German (de)
English (en)
Other versions
EP0754764B1 (fr
EP0754764A3 (fr
Inventor
Hermann Dr. Leying
Matthias Dr. Hinzpeter
Hans-Peter Dr. Fritton
Heiko Wittor
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
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Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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Publication date
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Publication of EP0754764A2 publication Critical patent/EP0754764A2/fr
Publication of EP0754764A3 publication Critical patent/EP0754764A3/fr
Application granted granted Critical
Publication of EP0754764B1 publication Critical patent/EP0754764B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the invention relates to a method and reagent or a kit for the specific determination of mRNA, i.e. of poly (A) sequences contained nucleic acids on coated solid phases.
  • mRNA can be quantified or detected both from mixtures of total RNA and in extracts from cell cultures and tissue cells. An upstream enrichment or isolation of the mRNA is not necessary.
  • mRNAs present in eukaryotic cells have a terminal sequence of approximately 20 to 250 adenosine nucleotides, which is used in most mRNA purification processes.
  • a standard method for the detection and quantification of mRNA today is the combined use of reverse transcriptase (RT) and PCR reaction (RT-PCR) (Larrick, JW, Trends Biotechnol. 10 , 146-152 (1992); Kawasaki, ES, PCR Protocols: A Guide to Methods and Applications (eds. Innis, MA et al.), Academic Press, San Diego, California (1990)).
  • RT reverse transcriptase
  • RT-PCR PCR reaction
  • RNA or mRNA Purified total RNA (rRNA, tRNA, mRNA) or purified mRNA is required as starting material for this method, since the enzymes used in RT-PCR, in particular polymerases, are very easily inhibited by impurities.
  • a number of methods are known for the purification of RNA or mRNA, such as, for example, extraction with organic solvents (for example phenol / chloroform), purification via oligo (dT) -cellulose or isolation using oligo (dT) -coated magnetic particles (Sambrook, J., Fritsch, EF and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2nd edition (1989); Farrell, RE, RNA Modologies: A Laboratory Guide for Isolation and Characterization, Academic Press (1993)).
  • the purification can moreover be carried out by hybridizing the poly (A) -bearing 3 'end of the eukaryotic mRNA with biotinylated oligo (dT).
  • the hybrid of biotin-oligo (dT) and mRNA can then be bound and isolated, for example, to magnetic particles coated with streptavidin.
  • biotin-oligo (dT) and mRNA can then be bound and isolated, for example, to magnetic particles coated with streptavidin.
  • An RT-PCR was then developed and described on a solid phase, ie on magnetic particles.
  • a disadvantage of this method is that after binding and washing the mRNA, the magnetic particles have to be converted into PCR-compatible, ie in particular heat-resistant, reaction vessels, so a further separation step is required.
  • the invention was therefore based on the object of providing an improved method for determining mRNA, as a result of which the disadvantages described in the prior art are overcome.
  • the mRNA is isolated and purified from the corresponding sample material in the vessel which is used for the reverse transcriptase (RT) reaction and the subsequent PCR reaction.
  • RT reverse transcriptase
  • Ie a so-called sample preparation (isolation and cleaning) and
  • RT-PCR Description or duplication (RT-PCR) are carried out in one and the same reaction vessel.
  • RNA total RNA
  • cell lysates fractions obtained from cell cultures
  • tissue cell extracts or tissue homogenates of human or animal origin and plant extracts the sample material to be analyzed comes into play Consideration.
  • Suitable lysis or hybridization buffers are based on buffer substances which have a good buffer capacity between a pH range of about 5 to 10, preferably between pH 7.0 and 8.5.
  • Corresponding buffer substances are, for example, Tris.HCl, HEPES, MOPS or Tris.Borate.
  • buffers suitable for the process according to the invention can contain a disulfide-reducing reagent, such as e.g. Dithioerythritol, dithiothreitol, mercaptoethanol, preferably contained in a concentration of 0.01 to 1% (w / v). It has also proven to be advantageous if the buffer system contains a denaturing substance, such as detergents, in relatively high concentrations.
  • a disulfide-reducing reagent such as e.g. Dithioerythritol, dithiothreitol, mercaptoethanol
  • the buffer systems which can be used for the process according to the invention can also contain further salts, such as e.g. Contain lithium chloride or other auxiliary substances. It has also proven to be advantageous if an RNase inhibitor, such as. B. a obtained from placenta, is present and / or the buffer solution was previously sterilized or decontaminated with dimethyl dicarbonate or diethyl pyrocarbonate.
  • oligo (dT) or oligo (U) nucleotide probes or corresponding peptide nucleic acid derivatives such as, for example, come as the labeled poly or oligonucleotide sample or labeled nucleotide derivative which carry a sequence complementary to poly (A).
  • PNA i.e. Nucleic acids with a peptide backbone of any length are considered. This is preferably 15 to 30 mer, often a 20 mer is sufficient.
  • All residues that can be coupled or bound to a specific matrix, such as haptens, proteins with an antigen or antibody structure, are suitable as a labeling group for the nucleotide sample.
  • Biotin which has proven to be particularly suitable here for example, bound to the oligo (dT) sequence via streptavidin or avidin to the solid phase, in the event that the organic chemical "coating substance" is streptavidin.
  • the coating substance can also be, for example, oligo (dT) nucleotides or suitable chelate compounds, which are suitable either for hybridization or complex formation for fixing corresponding samples.
  • the immobilization of mRNA on streptavidin-coated solid phases or reaction vessels thus represents a convenient and reliable method for mRNA extraction for the subsequent transcription and amplification.
  • other marker groups can also be used for fixing to the solid phase.
  • the nucleotide probes such as oligo (dT) probes, are produced by known processes or can be obtained from relevant suppliers.
  • the sample material containing polyadenylated mRNA is combined with the aqueous solution containing, for example, the labeled oligo (dT) sample.
  • the hybridization process is preferably carried out at approximately 37 ° C. and is usually completed after only a few seconds. A period of 10 seconds to 10 minutes, preferably 5 minutes, has been found to be sufficient for the hybridization in most cases.
  • An aliquot of the biotin-oligo (dT) -mRNA hybrid solution is then added directly to the coated, heat-stable reaction vessel.
  • the volume of the respective coated aliquot is optimally in the order of magnitude of the respective vessel volume and should not contain more than about 5 pmol of the hybrid.
  • a sample of approx. 20 to 200 ⁇ l of the lysate is sufficient.
  • For the immobilization on the coated solid phase there are already a few seconds, i.e. approx. 10 seconds incubation time at approx. 4 ° to 50 ° C, preferably sufficient at approx. 37 ° C in most cases, for safety's sake the process is maintained for one to 10 minutes in most cases.
  • a typical mixture for the RT reaction includes, in addition to the usual buffer substances and salts, magnesium ions, all four deoxynucleotide triphosphates (dNTPs), an enzyme with RT activity or an enzyme with RT and heat-stable DNA polymerase activity, such as DNA polymerase from Thermus thermophilus (Tth) and advantageously an RNase inhibitor.
  • dNTPs deoxynucleotide triphosphates
  • Tth Thermus thermophilus
  • RNase inhibitor an enzyme with RT activity
  • An enzyme with RT activity such as DNA polymerase from Thermus thermophilus (Tth) and advantageously an RNase inhibitor.
  • AMV- or M-MuLV-RT in particular in their RNaseH - form (US 5,244,797), are used as the enzyme with RT activity, preferably in a concentration of 0.01 to 5 U / ⁇ l.
  • the final concentration of the RT is preferably 0.1 to 1.0 U / ⁇ l.
  • RTs from other virus types or sources can also be used according to the invention.
  • the incubation for the cDNA synthesis can be carried out at approximately 30 ° to 75 ° C., preferably at 35 ° to 45 ° C., and is usually 30 to 120 minutes.
  • An incubation temperature of approx. 42 ° C has proven to be particularly advantageous in many cases.
  • reaction solution is removed and washed again thoroughly with washing buffer.
  • a specific volume of a PCR mix is then added to the coated reaction vessel, usually 20 to 200 ⁇ l and incubated under conditions that are not very stringent.
  • An optimal PCR mix includes a suitable buffer system, e.g. that in Maniatis et al.
  • PCR reaction itself takes place according to the measures known to those skilled in the art (so-called standard PCR), such as a first cycle at 95 ° C (5 min), 50 ° C (2 min) and 72 ° C (3 min); the subsequent cycles (up to a total of approx. 40 cycles) then take place according to the following mode: 95 ° C (1 min.), 50 ° C (2 min.) and 72 ° C (3 min. or 10 min. in the last one) Pass).
  • standard PCR such as a first cycle at 95 ° C (5 min), 50 ° C (2 min) and 72 ° C (3 min); the subsequent cycles (up to a total of approx. 40 cycles) then take place according to the following mode: 95 ° C (1 min.), 50 ° C (2 min.) and 72 ° C (3 min. or 10 min. in the last one) Pass).
  • the solution containing the transcribed and amplified DNA is removed from the reaction vessel and, for example, electrophoretically separated and, e.g. using ethidium bromide.
  • a further advantageous embodiment is that the RT and PCR reactions do not take place sequentially, but without an intermediate washing step in a coupled reaction. In such a simultaneous embodiment, the RT solution does not have to be removed after the RT reaction has ended.
  • a particularly advantageous embodiment is when a bifunctional enzyme which has both RT and DNA polymerase activity and is heat-stable is used. In such a so-called 1-pot / 1-buffer embodiment, an incubation time of approximately five minutes to one hour has proven to be particularly advantageous.
  • the DNA mixture obtained according to the invention is generally detected after electrophoretic separation using suitable hybridization methods (Southern blot, ELISA). However, it is also possible to directly detect the DNA mixture without prior separation. Appropriate methods are known to the person skilled in the art. Thus, according to the invention, mRNA could still be detected in less than 1 cell.
  • kits which instead of the two mixtures (d) and (e) comprises a mixture (d ') which contains an enzyme with RT and heat-stable DNA polymerase activity.
  • the features disclosed in connection with the method according to the invention apply to the kit.
  • streptavidin-coated PCR tubes are used to carry out the RT-PCR directly from cells without prior isolation of the RNA.
  • the cells are lysed in a lysis or hybridization buffer, the DNA is sheared, and the mRNA is immobilized in the reaction vessel by hybridizing the poly A + tail with BidT 20 and binding it to the streptavidin matrix.
  • the corresponding cDNAs are synthesized using reverse transcriptase and the specific transcripts are amplified using specific primers using PCR, followed by quantification, for example in agarose gel, using ethidium bromide staining.
  • This example describes the implementation of an RT-PCR directly from human K562 cells for the detection of mRNA of G3PDH.
  • DMDC dimethyl dicarbonate
  • DEPC diethyl pyrocarbonate
  • RNA 50 .mu.l of RT mix were pipetted into each of the individual vial places and incubated at 42.degree. C. for about 2 hours.
  • a vessel site with the highest RNA concentration was provided with RT Mix without reverse transcriptase; as a further control, a vessel site without RNA was provided with RT Mix with RT. This is followed by heat denaturation of the RT for 10 minutes at 65 ° C. and was washed 4 times with 250 ⁇ l wash buffer.
  • PCR products were separated in a 2% agarose gel (with ethidium bromide) at 100 V for 35 minutes and analyzed on the screen.
  • ⁇ -actin mRNA was determined. As a result, ⁇ -actin mRNA could be detected in less than one cell.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)
EP96111586A 1995-07-21 1996-07-18 Procédé et réactif pour la détermination spécifique d'ARNm Expired - Lifetime EP0754764B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19526431A DE19526431A1 (de) 1995-07-21 1995-07-21 Verfahren und Reagenz zur spezifischen Bestimmung von mRNA
DE19526431 1995-07-21

Publications (3)

Publication Number Publication Date
EP0754764A2 true EP0754764A2 (fr) 1997-01-22
EP0754764A3 EP0754764A3 (fr) 1999-04-21
EP0754764B1 EP0754764B1 (fr) 2003-04-09

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EP96111586A Expired - Lifetime EP0754764B1 (fr) 1995-07-21 1996-07-18 Procédé et réactif pour la détermination spécifique d'ARNm

Country Status (7)

Country Link
US (1) US5955271A (fr)
EP (1) EP0754764B1 (fr)
JP (1) JPH0928398A (fr)
AT (1) ATE236995T1 (fr)
CA (1) CA2181417C (fr)
DE (2) DE19526431A1 (fr)
ES (1) ES2193215T3 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1075542A4 (fr) * 1998-04-07 2004-08-18 Incyte Pharma Inc Dosages quantitatifs par hybridation de micro-arrangements

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT414209B (de) * 2000-03-17 2006-10-15 Greiner Bio One Gmbh Sammelgefäss für flüssigkeiten
US20030098271A1 (en) * 2001-11-26 2003-05-29 Ralph Somack Capsule and tray systems for combined sample collection, archiving, purification, and PCR
CN1316036C (zh) * 2003-01-06 2007-05-16 徐定邦 一种以聚合酶链式反应为基础的基因定量方法
EP1631668A1 (fr) * 2003-06-06 2006-03-08 Applera Corporation Dispositif de purification d'acide ribonucleique en larges volumes, et procede associe
US20110287951A1 (en) * 2009-01-30 2011-11-24 Emmert-Buck Michael R Methods and systems for purifying, transferring, and/or manipulating nucleic acids

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5354657A (en) * 1988-01-12 1994-10-11 Boehringer Mannheim Gmbh Process for the highly specific detection of nucleic acids in solid
IE64286B1 (en) * 1988-05-25 1995-07-26 Boehringer Mannheim Gmbh Process for the determination of an immunologically detectable substance and a suitable vessel therefor
GB8827160D0 (en) * 1988-11-21 1988-12-29 Apothekernes Lab Detection & quantitative determination of rna & dna
ATE118824T1 (de) * 1989-03-10 1995-03-15 Amoco Corp Immobilisierte oligonukleotidsonden und ihre verwendungen.
KR920700360A (ko) * 1989-03-22 1992-02-19 하리크 프리드리히 미끄럼 베어링
WO1993009250A1 (fr) * 1991-11-01 1993-05-13 Adelaide Children's Hospital Procede d'amplification en phase solide
MX9300492A (es) * 1992-01-29 1994-07-29 Hitachi Chemical Co Ltd Soporte inmovilizado de nucleotido y metodo para su produccion.
US5424189A (en) * 1993-03-05 1995-06-13 Kansas State University Research Foundation Bovine respiratory syncytial virus detection and primers
JPH06261800A (ja) * 1993-03-10 1994-09-20 Hitachi Ltd Ｒｎａウィルスの検出方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1075542A4 (fr) * 1998-04-07 2004-08-18 Incyte Pharma Inc Dosages quantitatifs par hybridation de micro-arrangements

Also Published As

Publication number Publication date
CA2181417C (fr) 2009-09-22
ES2193215T3 (es) 2003-11-01
DE19526431A1 (de) 1997-01-23
CA2181417A1 (fr) 1997-01-22
US5955271A (en) 1999-09-21
ATE236995T1 (de) 2003-04-15
DE59610324D1 (de) 2003-05-15
EP0754764B1 (fr) 2003-04-09
EP0754764A3 (fr) 1999-04-21
JPH0928398A (ja) 1997-02-04

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